Perez-Riverol Amilcar, Musacchio-Lasa Alexis, Fernandes Luis Gustavo Romani, Dos Santos-Pinto Jose Roberto Aparecido, Esteves Franciele Grego, Bazon Murilo Luiz, Zollner Ricardo de Lima, Palma Mario Sergio, Brochetto-Braga Márcia Regina
1Center for the Study of Social Insects, Department of General and Applied Biology, University of Sao Paulo State (UNESP/RC), São Paulo, Brazil.
2Center for Genetic Engineering and Biotechnology. Biomedical Research Division, System Biology Department, Ave. 31, e/ 158 and 190, Cubanacan, Playa, P.O. Box 6162, 10600 Havana, Cuba.
3 Biotech. 2020 May;10(5):217. doi: 10.1007/s13205-020-02202-8. Epub 2020 Apr 27.
Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate-phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in . The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients ( = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for venom allergy.
磷脂酶A1（PLA1）是在巴西东南部一种具有临床相关性的黄蜂（膜翅目：胡蜂科）毒液中鉴定出的三种主要过敏原之一。这种过敏原的重组形式（rPoly p 1）可用于开发毒液过敏的分子诊断方法。早期尝试使用BL21（DE3）细胞生产rPoly p 1时，不溶性rPoly p 1产量很高，但可溶性蛋白回收率较低（12%）。在此，我们旨在通过测试不同的表达条件、包涵体的溶解和蛋白质纯化来提高rPoly p 1在大肠杆菌中的产量。结果表明，在16℃和0.1 mM IPTG条件下表达可提高rPoly p 1的产量，其仍为不溶性形式，但在25℃下与含0.15 M NaCl、6 M尿素、pH 2.6的柠檬酸盐-磷酸盐缓冲液孵育2小时后，可溶性蛋白产量较高。该毒液过敏原也被克隆到pPICZαA载体中，以便在毕赤酵母X-33细胞中作为分泌蛋白进行可溶性表达，在培养上清液中产量几乎检测不到（纳克级）。相比之下，在大肠杆菌中改进生产后，可溶性和纯化的rPoly p 1产量提高了7倍（每升发酵液1.5 g）。用抗His抗体和质谱确认了该蛋白的身份。用40名过敏患者的血清进行免疫印迹评估了过敏原特异性IgE（sIgE）介导的识别。此外，rPoly p 1显示出高水平的诊断敏感性（95%）。本文所述的rPoly p 1生产优化策略，将为后续蛋白质复性、免疫学表征步骤提供所需的过敏原量，并最终用于开发黄蜂毒液过敏的分子诊断方法。
